Steam sterilization of autoclavable pelleted rodent diets causes increased or excessive pellet hardness which may result in prolonged intervals between litters, reduced weight gain, and reduced litter size. Very little is known about the optimum physical characteristics of rodent diets. The increase in pellet hardness due to steam sterilization and procedures for measuring rodent diet pellet hardness have not been published. A Chatillon Model TCM-200 test stand with exchangeable flat horizontal or concave receptacle bases and a DFI-200 gauge load cell with multiple types of upper exchangeable test jaws (large round-flat, medium round-flat, chisel, bullet, and cone shaped) were compared using preautoclaved and autoclaved NIH-31 rodent diet pellets to determine which type of hardness testing system would give the most accurate and reproducible results for measuring pellet hardness. The type and size of the contact area of the upper jaws significantly affected the force required to break the pellets. Significant differences were observed between the flat-horizontal and concave receptacle bases in the force required to break the pellets using the two round-flat upper jaws. In contrast, similar results were obtained with both bases when the bullet, chisel, or cone type upper jaws were used. Autoclaved pellets were 69.4% (range 49% to 94%) harder than preautoclaved pellets. These results suggest that different testing systems can be used for measuring pellet hardness, and that a standard procedure must be used in order to compare pellet hardness results between different testing laboratories. It was concluded that the flat-horizontal base and the larger round-flat end upper jaw gave the most reproducible results for measuring pellet hardness.